The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1β) with the luminal domain of IRE1β deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1β/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1β/α chimera. In vitro, AGR2 actively de-stabilised the IRE1β luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1β-AGR2 couple suggest that active repression of IRE1β by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

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REVIEW
This manuscript by Neidhardt et al focuses is directed towards defining the mechanism of IRE1beta activation.The establish a novel chimeric system where the luminal domain of endogenous IRE1alpha in CHO cells is replaced with the luminal stress sensing domain of IRE1beta.They found that overexpresson of the mucin chaperone AGR2 suppressed this constitutive activation of the IRE1alpha/beta chimera in cells and disrupted dimerization of the luminal domain in vitro.Further, they demonstrated that overexpression of the AGR2 substrate mucin could 'reactivate' IRE1alpha/beta chimeras, indicating that the interaction between the luminal domain of IRE1beta and AGR2 functions to sense specific stresses linked to mucin production.This manuscript, especially in context of its accompanying manuscript by Cloots et al, provides significant new insight into the regulation of IRE1beta in specialized cells such as goblet cells.The experiments are well performed and convincing, especially with the combination of efforts across the two co-submitted papers.I have no specific issues with any of the experimental designs or conclusions, all of which seem well performed and analyzed.I only have a couple simple suggestions that would further highlight the specificity and importance of the model described in this manuscript, which are included below.However, as indicated above, this manuscript and the accompanying manuscript provide very nice complementary evidence that AGR2 functions as a repressor of IRE1beta activity through engagement with the IRE1beta luminal domain and represents a key factor to sense specific proteostasis stresses within goblet cells (e.g., mucin misfolding/misfolding).

COMMENTS.
1.The authors are well positioned to address the potential contributions of BiP to the regulation of IRE1beta using their reporter cells expressing IRE1alpha/beta.Including some experiments where BiP is overexpressed in thse cells could reveal some potential cooperation between AGR2 and BiP in regulating IRE1beta activity through engaging the luminal domain.Either way, this result will not significantly impact the main conclusions of the study showing the importance of AGR2 in regulating IRE1beta activity, but they could demonstrate a more nuanced coordination between AGR2 and BiP in regulating IRE1beta activity in response to different types of stresses.This is alluded to in the text and seems like something that could be explored a bit more in this manuscript using the very nice tools established.2. Along the same lines, it could be useful to overexpress a more canonical PDI (e.g., PDIA1 or PDIA6 both of which have been implicated in regulating IRE1alpha) in the reporter cells to demonstrate the specificity of IRE1alpha/beta repression for AGR2.3. Inclusion of some immunopurifications of the IRE1alpha/beta-AGR2 interactions in cells, especially as it relates to the mucin experiments, would help support the model described within the manuscript.Notably, one would predict that overexpression of mucin should deplete AGR2 from IRE1alpha/beta, which could be reflected by reduced interactions observed by IP.These experiments would provide additional evidence to support the idea that IRE1beta-AGR2 interactons is a specific stress sensor of mucin folding load within the ER .

Referee #2:
This study demonstrates the mechanism of activation of IRE1beta in a context recapitulating molecules expressed in goblet cells.The authors show that the chaperone AGR2 binds to the IRE1 Ld domain, resulting in the inactivation of the protein.However, the presence of mucins relieves this interaction, leading to the engagement of the IRE1 cytosolic response.Importantly, the deficiency of AGR2 leads to the constitutive activation of IRE1beta but not IRE1alpha.On the other hand, the induction of misfolded proteins promotes IRE1alpha activation in the presence of ARG2 without allowing the activation of IRE1beta.Overall, this study and the accompanying paper support the model that IRE1s act as guardians of proteostasis in mammals rather than directly detecting specific ligands.They sense different insults and are released from inhibition by their specific sensor chaperones: AGR2 for IRE1beta and BiP for IRE1alpha.This is a fundamental and important discovery in the cell homeostasis maintenance field.
The strength of this manuscript relies on the biochemical approaches that nicely describe the molecular complex involved.The technical comments below are aimed at strengthening the central message of the paper.Some of the comments may have been addressed in the accompanying paper (cited in the introduction and in the discussion).1) While overall, the study is convincing, the fact that the chimeric construct (IRE1beta luminal domain and IRE1alpha cytosolic region) is constitutively active, as reported in Figure 1, could also be caused by conformational perturbation of fusing two proteins and/or from two different species mouse and chinese hamster.The study of a cell line with the entire alpha locus replaced with the entire locus IRE1beta locus could provide excellent additional controls.
2) The study relies on a model line recapitulating proteins expressed in goblet cells.However, using a cellular model expressing IRE1beta and AGR2 could strengthen the physiological relevance of the study.In that model, it would be possible to delete AGR2 and confirm that no specific IRE1beta ligands are expressed under physiological expression.
3) I could not find the nature of the cells used for the transfections-IP in Figure 3. Are those CHO cells deficient for endogenous IRE1 alpha?Overexpression of chaperones has the tendency to generate artifacts due to the plasticity of the folds that can be expressed upon overexpression.If technically feasible, I suggest the authors confirm these experiments using the stable lines described in Figure 1 and the AGR2 ko described in the appendix as controls.Moreover, the parallel comparison of both BiP and AGR2 binding to both IRE1 alpha and IRE1 beta would provide excellent controls that could support the possible scenario: IRE1 alpha is BiP controlled and beta AGR2 controlled.
4) The KEN-mediated differences observed in Figure 8 are difficult to interpret.These differences in XBP1s signaling could be due to slight structural imperfections caused by the chimera in the construct with the beta intracellular portion.Because the LD and transmembrane region of alpha is conserved in both cases, it is difficult to exclude that transmission of the signal is more effective when the alpha region LD is fused to the alpha KEN region than when the alpha region LD is fused to the beta KEN region.A possible additional experiment that would support the finding would be to repeat the experiment using the beta LD domain and monitoring constitutive activation, for example.If the beta/beta construct is less active than the beta/alpha construct, it could support the idea that the nature of the response is different.

Referee #3:
The unfolded protein response (UPR) is a homeostatic response of eukaryotic cells to cope with protein folding stress in the endoplasmic reticulum (ER).The central regulator of one of the three UPR branches is Ire1, a kinase and endonuclease that activates the transcription factor XBP-1 and cleaves a number of other mRNAs.Essential for Ire1 activation is the monomer-tooligomer transition.How the monomer-oligomer equilibrium of Ire1 is regulated by accumulating misfolded proteins in the ER is debated since many years.One model proposes that misfolded proteins bind directly to Ire1 and promote Ire1 oligomerization thereby acting as positive regulator.A second model poses that Ire1 spontaneously dimerizes and that the Hsp70 chaperone of the ER, BiP, actively dissociate Ire1 dimers/oligomers thereby acting as negative regulator.Since misfolded proteins bind to BiP and thus titrate BiP away from Ire1, according to the second model, deconvolution of direct and indirect effects of misfolded proteins is far from trivial.In mammalian cells there are two isoforms of Ire1, Ire1α the major isoform expressed in most cells and induced by general protein misfolding in the ER and Ire1β the isoform that is only expressed in some mucus producing cells, like goblet cells of the intestine, and induced by folding defects of mucins.To deconvolute the action of the Ire1 inducer, the authors exchanged the luminal domain of Ire1α with the luminal domain of Ire1β creating the Ire1β/α chimera in CHO cells where full-length Ire1β is not present.The chimeric Ire1β/α protein was constitutively active as demonstrated by several orthologous assays.Coexpression of the mucin specific chaperone AGR2 repressed the activity and this repressive action is counteracted by the coexpression of the AGR2 substrate MUC2.The authors show by biolayer interferometry that AGR2 binds to the luminal domain of Ire1β but not Ire1α.Using several orthologous in vitro assays with purified proteins including size exclusion chromatography, sedimentation velocity analytical ultracentrifugation and mass photometry, the authors demonstrate that the luminal domain of Ire1β is in monomer-dimer equilibrium and that AGR2 shifts the equilibrium to the monomeric state.Using a bioluminescence resonance energy transfer (BRET) assay, the authors demonstrate that AGR2 directly binds to Ire1β luminal domain dimers and induce monomerization.This seems to be an active process as monomerization rates in the presence of AGR2 are higher than in the presence of an excess of unlabeled Ire1β protein and increases with increasing AGR2 concentrations.The authors generate a number of mutants of AGR2 that have reduced ability to monomerize Ire1β in vitro and a diminished repressive effect on Ire1β/α activity in CHO cells.This study demonstrates beyond any reasonable doubt that (1) Ire1β is spontaneously dimerizing/oligomerizing in vivo and constitutively active in the absence of its negative regulator, the molecular chaperone AGR2; (2) Ire1β dimers are monomerized by AGR2; (3) amino acid replacements that reduce or prevent binding of AGR2 to Ire1β inactivate the monomerization activity of AGR2 in vitro and its repression of Ire1β activity in vivo.In my opinion this is an outstanding study, experiments are thoroughly designed and meticulously performed, interpretation of the data is cautious and rigorous, and conclusions seem fully warranted.There are only a few minor comments.

Minor comments:
In expanded view Fig. EV3 the authors present elution profiles of size exclusion chromatography for IRE1β in the absence of any interacting proteins or in the presence of different AGR2 wild type and mutant proteins.Normally the elution of column chromatography is given in volume units not time.It is curious that in panels A and B the time scale is 7 to 13 minutes whereas in panels C-E it is 7 to 13 seconds.The latter seems to be a typographic error.
Fig. 6E the authors present the increase in IRE1β dissociation rates with increasing AGR2 concentrations and fit the data to a one site binding equation with the dissociation rate of IRE1βn dimers in the absence of AGR2 as background.They give the K1/2 max in units of µM*s-1.This is not correct.The resulting K1/2 max will be in units of µM, because in the denominator of the equation for the one site binding reaction there is only KD + [Ligand] both of which should have the same units (mol/l).

Re: Manuscript EMBOJ-2023-114737 -Decision
Dear Editor, Thank you for your letter of 01-Aug-2023 detailing the reviewer's critiques of our paper.Thanks too to the reviewers for their time and effort.We are pleased by your invitation to submit a revised version addressing these criticisms.The following outlines a summary as well as a point-by-point list (with the reviewer's comments and our response) to the critiques articulated in your letter.Changes made in the manuscript are marked in green in the revised manuscript .docxformatted version.

Summary
The reviewers accepted the key conclusion of the study that the mucin chaperone AGR2 is a physiologically significant and selective repressor for the beta isoform of IRE1.They suggested experiments to further probe the specificity of the repressive action of AGR2 on IRE1 signalling by comparing it to a broadly-expressed reference protein of the same PDI family.Following this proposal, we transiently transfected PDIA1, previously implicated in IRE1 regulation (Yu et al., 2020) into IRE1/ expressing dual UPR reporter cells.Compared to AGR2 co-expression, presence of PDI1 failed to repress the deregulated IRE1/ chimera (Fig 2D of the updated manuscript).This experiment reinforces the existence of a special relationship between AGR2 and IRE1.
The reviewers also noted the potential to address the contribution of ER Hsp70 chaperone BiP to IRE1 signalling by employing our reporter cells.In line with these suggestions, we introduced SubA, a protease that inactivates BiP by cleaving its interdomain linker (Paton et al., 2006) into the reporter cells.As noted previously SubA-mediated BiP depletion strongly induced both IRE1/ signalling and the PERK dependent CHOP::GFP reporter (Amin-Wetzel et al., 2019), IRE1/ was less responsive to this intervention (Fig EV5A of the updated manuscript).In a reciprocal experimental design, BiP overexpression attenuated both, active IRE1/ (in absence of AGR2) and IRE1/ (upon ER stress induction).Together this suggests that whilst AGR2 selectively serves as a repressor for the  isoform, BiP governs IRE1 signalling and has the potential to contribute to IRE1 repression.In line with this notion, Bertolotti et al. (Bertolotti et al., 2000) (Fig. 3E, therein) showed that BiP co-IPs with IRE1 in a stress-dependent manner.
A circuit involving endogenous AGR2 and MUC2 has been implicated in regulating IRE1 (in the accompanying manuscript by Cloots et al.) and has been reconstructed here by co-expression of MUC2 and AGR2 using the fluorescent reporters in cells.The reviewers suggested to complement these findings with co-immunoprecipitation (IP) experiments to test the prediction that abundance of an IRE1-AGR2 complex would correlate negatively with the mucin load in the ER.We accept the logic behind this suggestion but note that we are unable to detect an IRE1/-AGR2 complex in cells expressing IRE1/ from the endogenous ERN1 locus.This likely reflects the low expression levels of the IRE1/ protein compounded by high off rates of the complex.This state of affairs all but preclude performing the experiment suggested by the reviewers in a system with a relevant concentration regime of IRE1/.
Elsewhere the reviewers suggested complementing the suite of cell lines employed in this study (IRE1/, IRE1/ and IRE1/) with cells expressing IRE1/ from the endogenous ERN1 locus.Whilst we recognise the theoretical utility of this approach, 16th Oct 2023 1st Authors' Response to Reviewers we note that creating such a cell line (via homologous recombination) is expected to take many months and therefore exceeds the time frame normally allowed for review.However, we have addressed the reviewer's suggestion editorially (see point-bypoint response below).

Point-by-point response
Reviewer 1: 1.The authors are well positioned to address the potential contributions of BiP to the regulation of IRE1beta using their reporter cells expressing IRE1alpha/beta.Including some experiments where BiP is overexpressed in these cells could reveal some potential cooperation between AGR2 and BiP in regulating IRE1beta activity through engaging the luminal domain.Either way, this result will not significantly impact the main conclusions of the study showing the importance of AGR2 in regulating IRE1beta activity, but they could demonstrate a more nuanced coordination between AGR2 and BiP in regulating IRE1beta activity in response to different types of stresses.This is alluded to in the text and seems like something that could be explored a bit more in this manuscript using the very nice tools established.
This point has been addressed experimentally.BiP levels were manipulated via two approaches: 1) Introduction of SubA, a protease that inactivates BiP by cleaving its interdomain linker (Paton et al. 2006) Bertolotti et al. 2000 Fig. 3E showing that BiP co-IPs with IRE1 in a stress-dependent manner.
2. Along the same lines, it could be useful to overexpress a more canonical PDI (e.g., PDIA1 or PDIA6 both of which have been implicated in regulating IRE1alpha) in the reporter cells to demonstrate the specificity of IRE1alpha/beta repression for AGR2.3. Inclusion of some immunopurifications of the IRE1alpha/beta-AGR2 interactions in cells, especially as it relates to the mucin experiments, would help support the model described within the manuscript.Notably, one would predict that overexpression of mucin should deplete AGR2 from IRE1alpha/beta, which could be reflected by reduced interactions observed by IP.These experiments would provide additional evidence to support the idea that IRE1beta-AGR2 interaction is a specific stress sensor of mucin folding load within the ER.

A circuit involving endogenous AGR2 and MUC2 has been implicated in regulating IRE1b (in the accompanying manuscript by Cloots et al.) and has been reconstructed here by co-expression of MUC2 and AGR2 using the fluorescent reporters in cells.
The reviewers suggested to complement these findings with co-immunoprecipitation (IP) experiments to test the prediction that abundance of an IRE1-AGR2 complex would correlate negatively with the mucin load in the ER.We accept the logic behind this suggestion but note that we are unable to detect an IRE1/-AGR2 complex in cells expressing IRE1/ from the endogenous ERN1 locus.This likely reflects the low expression levels of the IRE1/ protein compounded by high off rates of the complex.This state of affairs all but preclude performing the experiment suggested by the reviewers in a system with a relevant concentration regime of IRE1/.

Reviewer 2:
1) While overall, the study is convincing, the fact that the chimeric construct (IRE1beta luminal domain and IRE1alpha cytosolic region) is constitutively active, as reported in Figure 1, could also be caused by conformational perturbation of fusing two proteins and/or from two different species mouse and chinese hamster.The study of a cell line with the entire alpha locus replaced with the entire locus IRE1beta locus could provide excellent additional controls.
In the accompanying manuscript Cloots et al. shows that the intact IRE1, when expressed at even low levels in a heterologous system is constitutively active and subject to AGR2 repression (Fig 3A,therein), features mirrored by the IRE1/ chimera used here.Thus, we deem it unlikely that a corruption arising in the chimera gives rise to misleading experimental observations.Therefore, whilst we recognise the theoretical utility of the reviewer's suggestion, we note that creating such a cell line (via homologous recombination) is expected to take many months and therefore exceeds the time frame normally allowed for review.However, we have addressed the reviewer's suggestion editorially.
2) The study relies on a model line recapitulating proteins expressed in goblet cells.However, using a cellular model expressing IRE1beta and AGR2 could strengthen the physiological relevance of the study.In that model, it would be possible to delete AGR2 and confirm that no specific IRE1beta ligands are expressed under physiological expression.3F-I.

This point was addressed in the accompanying manuscript Cloots et al Figure
3) I could not find the nature of the cells used for the transfections-IP in Figure 3. Are those CHO cells deficient for endogenous IRE1 alpha?Overexpression of chaperones has the tendency to generate artifacts due to the plasticity of the folds that can be expressed upon overexpression.If technically feasible, I suggest the authors confirm these experiments using the stable lines described in Figure 1 and the AGR2 ko described in the appendix as controls.Moreover, the parallel comparison of both BiP and AGR2 binding to both IRE1 alpha and IRE1 beta would provide excellent controls that could support the possible scenario: IRE1 alpha is BiP controlled and beta AGR2 controlled.
We share the reviewer's concern regarding interpretation of experiments in which chaperones are overexpressed.Therefore to study the role of BiP, we emphasised its inactivation: Introduction of SubA, a protease that inactivates BiP by cleaving its interdomain linker (Paton et al. 2006)

strongly activated IRE1/ signalling and the PERK dependent CHOP::GFP reporter. In contrast, IRE1/ appeared less subordinate to SubA-mediated BiP depletion (Fig EV5A of the updated manuscript). The conclusion drawn from manipulation of endogenous BiP levels were also supported by the observation that when overexpressed, BiP served as a repressor for both active IRE1/ (in absence of AGR2) and IRE1/ (upon ER stress induction) (Fig EV5D of the updated manuscript). Overall, these findings highlight
that whilst AGR2 selectively represses the  isoform, BiP is more promiscuous and likely to partially contribute to IRE1 regulation.This is in line with Bertolotti et al. 2000 (Fig 3E, therein) showing that BiP co-IPs with IRE1 in a stress-dependent manner.
The concern voiced by the reviewer also applies to IRE1 and AGR2.However, as noted in the updated text we were unable to recover a complex between AGR2 and the low-abundant IRE1/ chimera by co-IP.In the accompanying paper, Cloots and colleagues document such a complex in colon tissue lysates (where expression levels of both partners are much higher, Fig 2E

therein). The analysis of IRE1-AGR2 complex in our paper was designed solely to test the role of mixed disulfide in complex formation, a question that when answered in the negative is unlikely to reflect an overexpression artefact (Fig 3C of the updated manuscript).
Elsewhere we have indicated the nature of the cells (wild-type CHO cells) used for IPs in Fig 3 .4) The KEN-mediated differences observed in Figure 8 are difficult to interpret.These differences in XBP1s signaling could be due to slight structural imperfections caused by the chimera in the construct with the beta intracellular portion.Because the LD and transmembrane region of alpha is conserved in both cases, it is difficult to exclude that transmission of the signal is more effective when the alpha region LD is fused to the alpha KEN region than when the alpha region LD is fused to the beta KEN region.A possible additional experiment that would support the finding would be to repeat the experiment using the beta LD domain and monitoring constitutive activation, for example.If the beta/beta construct is less active than the beta/alpha construct, it could support the idea that the nature of the response is different.
These are valid points.However, the main conclusion we draw from the comparison of the IRE1/ and IRE1/ chimera is their rather similar RIDD and XBP1 splicing activity.Thus, whilst both IRE1 isoforms are affected by divergent regulatory machineries, we found only modest differences comparing IRE1 KEN vs IRE1 KEN downstream signalling in our experimental system.These observations suggest that the driver for diversification in IRE1 function mainly arose from a specialisation in regulatory functions rather than effector functions.These considerations, together with the very significant effort posed by creating more knock-in cell lines, have led us to address this comment editorially.

Reviewer 3:
In expanded view Fig. EV3 the authors present elution profiles of size exclusion chromatography for IRE1β in the absence of any interacting proteins or in the presence of different AGR2 wild type and mutant proteins.Normally the elution of column chromatography is given in volume units not time.It is curious that in panels A and B the time scale is 7 to 13 minutes whereas in panels C-E it is 7 to 13 seconds.The latter seems to be a typographic error.
We thank the reviewer for this comment and have corrected this typographic error in the figures.The analytical SEC experiments have been performed consistently at a constant flow rate of 0.3ml/min and therefore elution volume can be derived from elution time.
Fig. 6E the authors present the increase in IRE1β dissociation rates with increasing AGR2 concentrations and fit the data to a one site binding equation with the dissociation rate of IRE1βn dimers in the absence of AGR2 as background.They give the K1/2 max in units of µM*s-1.This is not correct.The resulting K1/2 max will be in units of µM, because in the denominator of the equation for the one site binding reaction there is only KD + [Ligand] both of which should have the same units (mol/l).
We thank the reviewer for noticing this and have corrected this in the figure.Thanks for trusting us to handle this really insightful study; and congratulations!Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
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Design
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below.Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).the assay(s) and method(s) used to carry out the reported observations and measurements.an explicit mention of the biological and chemical entity(ies) that are being measured.an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

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Fig 2D of the updated manuscript.Whilst AGR2 expression repressed the deregulated IRE1/, PDIA1 did not.
to inform you that your manuscript has been accepted for publication in the EMBO Journal.
10) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online (see examples in https://www.embopress.org/doi/10.15252/embj.201695874).A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure strongly activated IRE1/ signalling and the PERK dependent CHOP::GFP reporter.In contrast, IRE1/ appeared less subordinate to SubA-mediated BiP depletion (Fig EV5A of the updated manuscript).2) When overexpressed, BiP served as a repressor for both active IRE1/ (in absence of AGR2) and IRE1/ (upon ER stress induction) (Fig EV5D of the updated manuscript).Overall, these findings highlight that whilst AGR2 selectively represses the  isoform, BiP is more promiscuous and likely to partially contribute to IRE1 regulation.This is in line with Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section)

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Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Checklist for Life Science Articles (updated January Study protocol Information included in the manuscript? In which section is the information available?
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsincludeclearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.(ReagentsandTools Table, Materials and Methods, Figures, Data Availability Section)If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.

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(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority

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and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (